The present invention relates to an improved method for the determination of lipid bound sialic acid and more particularly to a method which is less time-consuming and requires a much smaller sample than the prior methods.
Much work has been done which indicates that elevated sialic acid content in blood sera of a patient is an indication of the presence of cancer.
Thus, for example, U.S. Pat. No. 4,146,603 to Davidson et al discloses and claims a fairly complex series of procedures whereby elevated sialic acid content is a determinant with respect to cancer specific determinations.
MacBeth and Bekesi, Cancer Res. 22, 1170-1176, 1962, measured plasma glycoproteins and found galactose and mannose values were seen in breast cases without metastases. Kloppel et al., 1977, Proc. Natl. Acad. Sc. 74, 3011-3013, reported 2.5-fold increases of serum sialic acid glycolipids in mice bearing transplantable mammary carcinomas and 2-fold increases in human carcinoma patients. The method involved column chromatographic separation of the gangliosides. A minimum of 1 ml whole blood was required. Kloppel et al., 1978, Am. J. Vet. Res. 39, 1377-1380, also reported increases of sialic acid in 92% of 24 dogs; however, a number of false positive were observed in dogs with other disorders. In leukemic AKR/J mice, Lengle, 1979, J. Natl. Cancer Inst. 62, 1565-1567, found increased lipid bound sialic acid in their plasma and thymic lymphocytes. Lipid bound sialic acid levels were found increased in plasma and erythrocytes of humans bearing melanomas (Portoukalian et al., 1978, Biochem. Biophys. Res. Commun. 85, 916-920). Chromatographic separation and purification on columns was followed by evaluation on chromatoplates. Silver et al., 1978, Cancer 41, 1497-1499; 1979, Cancer Res. 39, 5036-5042, have reported elevated serum sialic acid values in melanoma patients that were significantly related to the tumor burden; however, 36% of patients with observable tumors showed no elevated serum sialic acid. Hogan-Ryan et al., 1980, Br. J. Cancer 41, 587-592, reporting on total bound serum sialic acid in patients with breast cancer found elevations that corresponded with tumor stage.
The specific method over which the present invention is an improvement is disclosed in the American Association for Cancer Research Annual Meeting PROCEEDINGS Vol 21, March 1980 as Abstract No. 728 by Katopodis et al. Briefly, this method requires that a 100 .mu.l plasma sample (reduced to 50 .mu.l) be extracted with 6 ml of 2 to 1, chloroform to methanol, volume to volume mixture. The lipid extract is then partitioned with 0.2 of its volume of water. The aqueous phase is evaporated to dryness and the residue redissolved in water. The lipid bound sialic acid is then purified by trichloroacetic acid-phosphotungstic acid precipitation and, after the removal of the supernatant from the resultant precipitate, the precipitate is determined by the Svennerholm and Miettien method (Svennerholm, Quantitative Estimation of Sialic Acid . . , Biochem. Biophys. Acta 24, pp. 604-611 (1957)).